1.  The exact mechanism of pertussis toxin secretion is still a puzzle.  It is known that the toxin must be fully assembled, but that a single mutation of arginine-57 to lysine in the S1 subunit dramatically decreased the secretion of pertussis toxin by the bacteria.  This will be used as a negative control for the binding assay. 

            A.  Affinity of pertussis toxin to one secretion protein

-General idea:  Pour Ptx over a column that has a pertussis toxin secretion system protein bound to it and look for interactions between the toxin    and the protein.

The first thing to do is to isolate the different Ptl proteins.  A His tag can be added to an individual Ptl protein gene, then each gene can be cloned into an expression vector and transformed into E. coli cells.  These cells are then lysed and the lysate is passed over a Nickel column.  The Ni-column will bind to the His-tagged protein.  Next, a wild type pertussis toxin is passed over the column.  The pertussis toxin is commercially available.  The presence of toxin in the flowthrough can be detected by SDS-PAGE and immunoblot analysis using antibodies against the S1 subunit of pertussis toxin.  If toxin is bound to the Ptl protein, it can be eluted off the column by EDTA, which disrupts the histidine-nickel interaction.  To determine if the interaction seen is relevant to secretion, the R57L mutant will be passed over the column to see if it will bind to the Ptl protein.  If the protein’s function is to bind toxin so it is secreted, then it will not bind the mutant toxin.  Another control is to use both N and C-terminal His tags to make sure the addition of the His tag is not disrupting the binding.

2.  It is known that PtlC and PtlH both have ATP binding domains, and that mutations in these domains stops the transport of pertussis toxin.  Whether these proteins act as kinases and signal the opening of the gate, if they function as ATPase and provide energy for the transport process, or if they function in some other way to transport the toxin is still unknown. 

B.     Function of PtlC and PtlH

-General idea:  Compare the expression of proteins in B. pertussis that are wild type for PtlC and PtlH, and bacteria that have mutations in the nucleotide binding domain of PtlC and PtlH. 

The point mutation K462R in the Walker box A region of ptlC has already been made using PCR mutagenesis.  In order to determine differences in protein expression between this mutant and the wild type, a 2-D gel and MALDI-TOF can be performed as well as a microarray assay.  To do the 2-D gel, the bacteria will be lysed by sonication and the extract run on a 2-D gel.  Any spots that appear different between the mutant and wild type box will be analyzed using MALDI-TOF to determine their identity.  If the gel is too cluttered, the proteins from different cellular fractions could be analyzed with the 2-D gel.  The bacteria can be treated with EDTA and lysozyme then centrifuged to isolate the cells.  The supernatant will contain the periplasmic proteins.  Next the bacteria can be osmotically lysed or lysed using a French press.  Spin down in an ultracentrifuge.  The supernatant will contain the cytosolic proteins and the pellet will contain the membrane proteins.  Each of these fractions can be run on a 2-D gel and the mutant and wild type gels can be compared.